Blotting techniques are widely used in biochemical analyses to identify regions of protein-antibody binding or nucleic acid-nucleic acid hybridization. Blotting techniques begin with the electrophoresis of macromolecules, either proteins or nucleic acids, on a solid support. Electrophoresis is a technique that depends on the relative mobility of charged particles, in this case macromolecules, under an electric field in a solid support. This support is usually an agarose slab gel or acrylamide slab gel. A solution containing the macromolecules of interest is loaded into one end of the slab gel. An electric field is created in the slab gel and causes the macromolecules to travel into the slab gel. The goal of electrophoresis in these cases is to create a distributional pattern of macromolecules within the solid support.
There are various ways that these macromolecules can be distributed. Macromolecules travel in an electric field with a speed proportional to the size of the molecule. In separating populations of molecules that have different sizes but carry similar charges, such as populations of RNA or DNA, the smaller molecules will travel farther on the solid gel support than the larger molecules. Therefore, the molecules separate on the slab gel on the basis of size.
Another way to separate molecules on a slab gel is on the basis of their charge. Populations of protein molecules differ as to the net charge that each molecule carries in a solution of given pH. If during the electrophoretic procedure a pH gradient is formed across the solid gel support, molecules will migrate into the gel until the molecules reach their isoelectric point in the pH gradient. At the isoelectric point, the molecule carries no charge. Because different protein molecules have different isoelectric points, populations of protein molecules can be distributed in the slab gel via the charge they carry.
It is possible to combine the two techniques of charge and size separation. The technique of two-dimensional gel electrophoresis involves two different electrophoretic separations of protein molecules. The molecules are separated in one dimension by their isoelectric point. The molecules are then separated in the second dimension according to their molecular size.
It is often advantageous to expose these separated macromolecules to other components and analyze the binding of components to the separated macromolecules. For example, a particular messenger RNA might have been isolated and cloned, and a scientist might be interested in isolating the gene corresponding to that messenger RNA. Analyzing which DNA fragment in a restriction enzyme digest of genomic DNA could hybridize to a cDNA clone created from this messenger RNA would facilitate the isolation of the gene.
The technique of Southern blotting allows one to analyze these DNA-DNA hybridizations. In a Southern blotting procedure, DNA fragments are first electrophoretically distributed within a slab gel. Typically, the slab gel is immersed in a basic solution that denatures the DNA fragments. The slab gel is then laid on a stack of buffer-soaked filter paper. A blotting medium, which will receive the macromolecules as they leave the slab gel, is placed against the gel. A stack of dry filter papers is placed on top of this blotting medium.
Capillary action draws the buffer from the buffer-soaked filter paper through the gel and through the blotting medium. The buffer flows into the dry filter papers stacked on top of the blotting medium. DNA fragments are transferred from the slab gel onto the blotting medium. The fragments stick to the blotting medium and are not transferred to the dry filter papers because the pore size of the blotting medium is such that small molecules pass through but larger macromolecules can be trapped. The blotting medium now contains a copy of the DNA distribution pattern of the slab gel. This macromolecule-laden blotting medium is called a "blot."
Blots can be created in ways other than through capillary action. Recently blots have been created by a process called "electroblotting." An electric field is created across the slab gel and blotting medium and the macromolecules are electrophoresed onto the blotting medium. This procedure is usually quicker than the capillary blotting procedure.
The blot is usually placed in a plastic pouch with a solution of radioactive probe, either DNA or RNA. Molecular hybridization will occur with those portions of the blotted DNA molecules that have a nucleotide sequence which is sufficiently complementary to the radioactive DNA or RNA probes. The blotting medium can then be washed, dried, and exposed to x-ray film. When the film is developed, it will retain an image of the hybridization pattern of the radioactive probe. Therefore, the particular fragment in a DNA digest that hybridizes with this particular probe can be identified via its location on this developed x-ray film.
In another example, electrophoretically separated proteins are often incubated with antibody-containing solutions, with the purpose of analyzing the position of the protein molecules that specifically bind to the antibodies. This technique is referred to as "Western blotting". Radiolabelled antibodies can reveal the location of antibody-protein binding. Additionally, locations of specific binding can be visualized in other ways. A common technique is to conjugate an identifiable enzyme to the antibody. For instance, peroxidase can be attached to the bound antibody. If one incubates this antibody-laden blot in a solution containing a peroxidase substrate and an oxidizable chromogen, the areas containing peroxidase will change color and will be easily visualized.
The blotting medium is typically a nitrocellulose filter, commercially available from many sources. Nitrocellulose filters are flat sheets made from cellulose nitrate, a nitric acid ester of cellulose. This filter binds DNA strongly, and the DNA can be permanently fixed to the nitrocellulose filter by baking at approximately 80.degree. C. Proteins and nucleic acids are adsorbed to the surface of nitrocellulose by electrostatic and hydrophobic interaction. Nitrocellulose filters are very widely used for blotting techniques, but they are mechanically fragile and expensive.
Nylon was introduced in blotting mediums because of its greater mechanical strength and higher protein binding capability. However, nylon membranes are not compatible with any protein stain except the biotin/avidin system. Recently, hydrophobic PVDF (polyvinylidene difluoride) membrane has been introduced. This membrane has mechanical strength, high binding capacity and good compatibility with most Western blot procedures. This material, however, is somewhat expensive and not without staining artifacts. For example, in our lab we have used PVDF membranes to blot milk proteins. We frequently find a white spot during immuno-staining. These white spots are an artifact of the PVDF membrane.
What is needed in the art of macromolecule blotting is a blotting medium that is easy to prepare, inexpensive, compatible with standard macromolecule visualization methods, and mechanically strong enough to withstand manipulation in blotting procedures.